结核分枝杆菌lhp-esat6融合基因穿梭表达载体的构建及在BCG中的表达

目的　构建能表达结核分枝杆菌早期分泌蛋白CFP10 -ESAT6融合蛋白的重组卡介苗 (recombinantBCG ,rBCG)。方法　以 pQE30 -CFP10 -ESAT6质粒为模板 ,通过PCR扩增 6 33bplhp -esat6基因 ,将该基因定向克隆到穿梭表达载体pJCH0 2中构建重组 pJCH0 2 -CFP10 -ESAT6质粒。用电穿孔法将重组质粒导入BCG菌构建rBCG ,将rBCG培养 2 3天 ,于收菌前 3天每天 4 5℃热诱导 4 5min ,对表达产物作SDS -PAGE及免疫印迹分析。结果　重组质粒 pJCH0 2 -CFP10 -ESAT6经酶切及测序证实构建成功 ,并在BCG中经热诱导成功表达出了具有CFP10及ESAT6抗原性的CFP10 -ESAT6融合蛋白。结论　成功构建能表达CFP10 -ESAT6融合蛋白的重组BCG ,为发展新型结核病疫苗奠定了基础。

Construction of the recombinant shuttle-plasmid with lhp-esat6 fusion gene of Mycobacterium tuberculosis and its expression in BCG

Objective To construct a recombinant fusion protein CFP10-ESAT6 of Mycobacterium tuberculosis expressed in BCG,the 633bp lhp-esat6 fusion gene was amplified by PCR from pQE30-CFP10-ESAT6 plasmid and then cloned into E.coli-BCG shuttle-plasmid pJCH02 to get pJCH02-CFP10-ESAT6.The recombinant plasmid was introduced into BCG by electroporation and the rBCG was cultured for 23 days,induced daily at 45℃ for 45min at the last three days.Those products from rBCG were analyzed by SDS-PAGE and Western blotting.It was found that the lhp-esat6 fusion gene was correctly inserted into the vector pJCH02 and it was confirmed by restriction endonuclease digestion and sequence analysis.The recombinant CFP10-ESAT6 fusion protein was identified for both antigenicity by Western blotting.It concludes that the rBCG expressing the CFP10-ESAT6 fusion protein has been successfully constructed and the results of this study could provide for the basis to develop a new tuberculous vaccine.